CDNA LIBRARIES PREPARED FROM PURE NEURONAL PROCESSES

该建议的具体目的是从神经元过程中创建cDNA文库，以研究mRNA在树突和/或轴突中的定位。 大量研究表明，mRNA在神经元过程中的定位和这些mRNA的调节翻译是神经元中基因表达的重要机制。 为了开始研究这种形式的基因表达，我们将创建分离的神经元过程的制剂，开发用于从这些制剂中收集的mRNA的严重有限的量创建cDNA文库的方法，并开发用于分析mRNA种类在基础和刺激状态下的神经元过程中的代表性和积累的方法。 我们将使用培养的感觉神经元和培养的小鼠海马神经元。 失活神经元的独特优势在于可以从培养的神经元中手动去除细胞体和神经胶质，留下纯的神经元突起作为cDNA文库构建的起始材料。 啮齿动物海马神经元可以在膜上培养，使得神经元轴突和树突可以穿透膜中的孔，因此可以与细胞体分离。 使用这些准备工作，我们将：1.开发纯神经元过程的制剂2.开发从这些神经元过程中创建cDNA文库的方法3.开发方法来测试的代表性和本地积累的转录在未受刺激和刺激的神经元过程。

The specific aim of this proposal is to create cDNA libraries from neuronal processes in order to study the localization of mRNAs to dendrites and/or axons. A number of studies have indicated that localization of mRNAs to neuronal processes and regulated translation of these mRNAs serves as an important mechanism of gene expression in neurons. To begin to study this form of gene expression, we will create preparations of isolated neuronal processes, develop methodologies for creating cDNA libraries from the severely limited amount of mRNA collectable from these preparations, and develop methodologies for analyzing the representation and accumulation of mRNA species in neuronal processes in both basal and stimulated states. We will use cultured Aplysia sensory neurons and cultured mouse hippocampal neurons. The unique advantage of Aplysia neurons is that cell bodies and glia can be manually removed from cultured neurons, leaving pure neuronal processes as a starting material for cDNA library construction. Rodent hippocampal neurons can be cultured on membranes such that the neuronal axons and dendrites can penetrate through the pores in the membrane and can therefore be separated from cell bodies. Using these preparations we will: 1. Develop preparations of pure neuronal processes 2. Develop methodologies for creating cDNA libraries from these neuronal processes 3. Develop methodologies to test the representation and localized accumulation of transcripts in unstimulated and stimulated neuronal processes.